UNIVERSITA' DEGLI STUDI DI PADOVA ___________________________________________________________________ SCUOLA DI DOTTORATO DI RICERCA IN SCIENZE DELLE PRODUZIONI VEGETALI INDIRIZZO PROTEZIONE DELLE COLTURE - CICLO XXIII Dipartimento di Agronomia Ambientale e Produzioni Vegetali Symbiotic and associated bacteria in Tephritid flies Direttore della Scuola : Ch.mo Prof. Andrea Battisti Supervisore : Ch.mo Prof. Vincenzo Girolami Dottoranda : Claudia Savio DATA CONSEGNA TESI 31 gennaio 2011 Declaration I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of the university or other institute of higher learning, except where due acknowledgment has been made in the text. January 31st, 2011 Claudia Savio A copy of the thesis will be available at http://paduaresearch.cab.unipd.it/ Dichiarazione Con la presente affermo che questa tesi è frutto del mio lavoro e che, per quanto io ne sia a conoscenza, non contiene materiale precedentemente pubblicato o scritto da un'altra persona né materiale che è stato utilizzato per l’ottenimento di qualunque altro titolo o diploma dell'università o altro istituto di apprendimento, a eccezione del caso in cui ciò venga riconosciuto nel testo. 31 Gennaio 2011 Claudia Savio Una copia della tesi sarà disponibile presso http://paduaresearch.cab.unipd.it/ Dichiarazione Questo lavoro di tesi è stato svolto grazie alla borsa di studio triennale finanziata da Veneto Agricoltura nell’ambito della convenzione con l’Università degli Studi di Padova. Dall’articolo 5 della Convenzione: Veneto Agricoltura potrà utilizzare, esclusivamente ai fini della propria attività di ricerca e sperimentazione, i risultati degli studi e delle ricerche svolte dai dottorandi. Potrà altresì citare nelle proprie pubblicazioni il contenuto delle tesi finali presentate dai dottorandi al termine di ciascun ciclo di dottorato, con l’obbligo di espressamente menzionare il titolo della tesi, l’autore, il tutore e la Scuola di dottorato. Citazione suggerita: Savio C., 2011. Symbiotic and associated bacteria in Tephritid flies. Supervisor: Prof. Girolami V. Scuola di Dottorato di Ricerca in Scienze delle Produzioni Vegetali. 4 Table of contents Table of contents ......................................................................................................... 5 Riassunto ...................................................................................................................... 9 Summary .................................................................................................................... 11 Chapter 1 - Introduction ................................................................................................ 13 Objectives and content of the thesis ......................................................................... 34 References .................................................................................................................. 35 Chapter 2 - Evidence of two lineages of the symbiont “Candidatus Erwinia dacicola” in Italian populations of Bactrocera oleae (Rossi) based on 16S rRNA gene sequence Introduction ................................................................................................................ 45 Materials and methods ............................................................................................... 47 Olive fly sampling .................................................................................................. 47 Insects dissection and symbionts analysis................................................................ 47 Insect host analysis ................................................................................................. 48 Sequencing and data analysis .................................................................................. 49 Results and Discussion ............................................................................................... 50 Erwinia dacicola diversity ....................................................................................... 50 Erwinia dacicola haplotypes distribution ................................................................ 52 Matching extent of symbiont and host haplotypes ................................................... 56 Chapter 3 - Distribution of olive fly symbiont lineages in Mediterranean populations of Bactrocera oleae (Rossi). Introduction ................................................................................................................ 63 Materials and methods ............................................................................................... 64 Samples collection .................................................................................................. 64 DNA extraction, amplification and sequencing ....................................................... 64 Candidatus Erwinia dacicola analysis ..................................................................... 65 Host data analysis ................................................................................................... 65 Results ........................................................................................................................ 66 5 Erwinia dacicola diversity and distribution ............................................................. 66 Bactrocera oleae mitochondrial diversity ................................................................ 70 Testing the association between Erwinia dacicola and B. oleae haplotypes ............. 75 Discussion ................................................................................................................... 76 Chapter 4 – Bacterial communities associated with Rhagoletis completa Cresson and Rhagoletis cerasi (Linnaeus) (Diptera: Tephritidae) Introduction ................................................................................................................ 83 Materials and methods ................................................................................................ 84 Rhagoletis cerasi: host collection and immature stages handling ............................. 84 R. cerasi and R. completa: adults stages collections and handling ............................ 85 DNA extraction, amplification and data analysis ..................................................... 86 Results and discussion ................................................................................................ 87 Rhagoletis cerasi immature stages ........................................................................... 87 Wild flies ................................................................................................................ 89 Conclusions ................................................................................................................ 94 Chapter 5 - Green fluorescent protein (GFP)-labeling of indigenous enterobacteria and persistence in their natural host Rhagoletis completa Cresson (Diptera, Tephritidae) ............................................................................................................... 95 Chapter 6 - Phylogenetic relationships between flies of the Tephritinae subfamily (Diptera,Tephritidae) and their symbiotic bacteria Introduction .............................................................................................................. 107 Materials and methods .............................................................................................. 110 Origin of the biological material ............................................................................ 110 Insects ................................................................................................................... 111 Symbionts ............................................................................................................. 114 Coevolutionary analysis ........................................................................................ 115 Results....................................................................................................................... 117 Insects ................................................................................................................... 117 Symbionts ............................................................................................................. 120 6 Coevolutionary analysis ........................................................................................ 121 Discussion .................................................................................................................122 Molecular relationships among the tribes of the subfamily Tephritinae ................. 122 Distribution of the symbiosis among the subfamily Tephritinae ............................ 124 Concordant evolution of subfamily Tephritinae with its symbiotic bacteria ........... 126 Conclusions .................................................................................................................. 141 References .................................................................................................................... 143 Aknowledgments .......................................................................................................... 159 7 8 Riassunto I Tefritidi, noti anche come “fruit fly”, rappresentano una vasta famiglia di Ditteri